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The Journal of Neuroscience, June 15, 1998, 18(12):4532-4547
Multivesicular Release at Single Functional Synaptic Sites in
Cerebellar Stellate and Basket Cells
Céline
Auger,
Satoru
Kondo, and
Alain
Marty
Arbeitsgruppe Zelluläre Neurobiologie, Max-Planck-Institut
für biophysikalische Chemie, 37077, Göttingen, Germany
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ABSTRACT |
The purpose of the present work was to test the hypothesis that no
more than one vesicle of transmitter can be liberated by an action
potential at a single release site. Spontaneous and evoked IPSCs were
recorded from interneurons in the molecular layer of cerebellar slices.
Evoked IPSCs were obtained using either extracellular stimulation or
paired recordings of presynaptic and postsynaptic neurons. Connections
were identified as single-site synapses when evoked current amplitudes
could be grouped into one peak that was well separated from the
background noise. Peak amplitudes ranged from 30 to 298 pA. Reducing
the release probability by lowering the external
Ca2+ concentration or adding Cd2+
failed to reveal smaller quantal components. Some spontaneous IPSCs
(1.4-2.4%) and IPSCs evoked at single-site synapses (2-6%) were
followed within <5 msec by a secondary IPSC that could not be
accounted for by random occurrence of background IPSCs. Nonlinear summation of closely timed events indicated that they involved activation of a common set of receptors and therefore that several vesicles could be released at the same release site by one action potential. An average receptor occupancy of 0.70 was calculated after
single release events. At some single-site connections, two closely
spaced amplitude peaks were resolved, presumably reflecting single and
double vesicular release. Consistent with multivesicular release,
kinetics of onset, decay, and latency were correlated to IPSC
amplitude. We conclude that the one-site, one-vesicle hypothesis does
not hold at interneuron-interneuron synapses.
Key words:
synaptic transmission; cerebellum; GABA; stellate cells; basket cells; quantal analysis; vesicular release
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INTRODUCTION |
Synaptic signals fluctuate because
of the random occurrence of discrete units ("quanta") reflecting
the exocytosis of single presynaptic vesicles (Katz, 1969 ). At central
synapses, amplitude histograms of evoked synaptic potentials have been
fitted with binomial distributions, and the number of peaks is believed
to correspond to the number of release sites (Redman, 1990). Such a
correspondence requires that a single release site contributes at most
one quantum, either because some intrinsic limitation prevents the
release of more than one vesicle (Triller and Korn, 1982) or because
the postsynaptic receptors are close to saturation so that simultaneous
release events are indistinguishable from singular events (Redman,
1990). The original formulation of the one-site, one-quantum hypothesis
and of its more restrictive variant the one-site, one-vesicle
hypothesis were based, however, on model-dependent interpretations of
amplitude distributions at multisite connections. Such methods are
indirect and have been subjected to criticism (Clements, 1991; Bekkers,
1994).
Recently, more direct evidence started to accumulate indicating that
indeed, single-site signals are limited to one quantum. At excitatory
synapses between CA3 pyramidal cells and interneurons in the
hippocampus, morphological evidence indicates predominantly single-site
contacts, whereas evoked postsynaptic signals display a Gaussian
amplitude distribution (Gulyàs et al., 1993; Arancio et al.,
1994). Likewise, some mossy fiber-granule cell synapses in the
cerebellum have single-Gaussian amplitude distributions and behave as
expected for single-site synapses when the the external Ca2+ concentration is lowered (Silver et al., 1996).
The striking absence of double-sized events at single-site synapses
(Gulyàs et al., 1993; Arancio et al., 1994; Silver et al., 1996)
leaves little doubt about the validity of the one-site, one-quantum
hypothesis. The question that remains open, however, is whether the
lack of a secondary peak reflects inhibition of multiple release
(one-site, one-vesicle hypothesis) or saturation of postsynaptic
receptors.
Tong and Jahr (1994) and Scanziani et al. (1997) reported a
differential block of EPSCs by low-affinity competitive antagonists under normal and high release probability conditions, indicating that
postsynaptic receptors are exposed to a higher or more prolonged neurotransmitter concentration in the latter case. Moreover, the time
course of synaptic currents is prolonged under high release conditions
in certain synapses (for review, see Barbour and Häusser, 1997).
Although these observations can be interpreted as indicating multivesicular release (Tong and Jahr, 1994), it was recently pointed
out that they are equally well explained by cross-talk between
neighboring synapses (Barbour and Häusser, 1997; Scanziani et
al., 1997).
The present work examines the question of multivesicular release by
taking advantage of the properties of interneuron-interneuron synapses
in the molecular layer of the cerebellum. At these synapses, miniature
currents are very large, with a mean of 140 pA at  60 mV under
symmetrical Cl concentration conditions (Llano and
Gerschenfeld, 1993). Furthermore, the mean size of spontaneous IPSCs
recorded in control solution is similar to that of miniature IPSCs
(mIPSCs), suggesting that some connections involve single release sites
(Llano and Gerschenfeld, 1993). We report here that multivesicular
release occurs in this preparation.
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MATERIALS AND METHODS |
Slice preparation. Cerebellar slices were prepared as
described previously (Llano et al., 1991). Briefly, rats (12- to
16-d-old) were killed by decapitation under anesthesia with Metofane.
The vermis of the cerebellum was quickly removed and placed in ice-cold solution. Slices, 180-µm-thick, were cut parallel to the sagittal plane. They were kept in a vessel bubbled with 95% O2 and
5% CO2 at 33°C for 1-6 hr before recording.
Patch-clamp recordings. Patch-clamp recordings were made
from inhibitory interneurons (stellate or basket cells) of the
molecular layer of the cerebellum. The cells were differentiated from
migrating granule cells or glia by the size of their soma (~8 µm).
The identification was confirmed by the observation of spikes in the
cell-attached mode and of spontaneous synaptic activity as well as of
voltage-dependent Na+ currents after breaking into
the cell. The tight-seal whole-cell recording technique was used in all
cases to record postsynaptic IPSCs. When filled with internal saline,
the recording pipettes had a resistance of 3-5 M . The internal
solution used was either a K+- or
Cs+-based solution. The K+-based
solution contained (in mM): 150 KCl, 4.6 MgCl2, 0.1 CaCl2, 1 EGTA, 10 K-HEPES, 0.4 NaGTP, and 4 NaATP, or alternatively 120 KCl, 4.6 MgCl2, 1 CaCl2, 10 EGTA, 10 K-HEPES, 0.4 NaGTP, and 4 NaATP. The Cs+-based
solution contained (in mM): 124 CsCl, 4.6 MgCl2, 0.1 CaCl2, 1 EGTA, 10 HEPES, 0.4 NaGTP, and 4 NaATP. The holding potential was 60 mV. The
mean cell capacitance and uncompensated series resistance were 5.9 ± 1.9 pF and 16.9 ± 7.3 M, respectively. The series
resistance was compensated between 50 and 90%.
The bath was perfused continuously at a rate of 1-1.5 ml/min with an
external solution of the following composition (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. In control recordings, the
external solution was supplemented with 2 mM
CaCl2 and 1 mM MgCl2. This was also
the solution used for cutting and keeping the slices. The probability
of release was decreased in some experiments by reducing the external
Ca2+ concentration down to 1.5-1 mM
Ca2+ and increasing the Mg2+
concentration to 1.5-2 mM Mg2+, or by
adding 2.5 µM Cd2+ to the control
solution. The external solution was bubbled continuously with a mixture
of 95% O2 and 5% CO2 to maintain the pH at
7.4. All experiments were performed at room temperature.
Extracellular stimulation. A presynaptic interneuron was
stimulated using an extracellular electrode. The electrode was made of
a patch pipette that was filled with extracellular saline and had a
resistance of 1-4 M. The ground electrode for the stimulation circuit was made with a platinum wire wrapped around the stimulation electrode. A voltage pulse (1-100 V) was applied through the
stimulation electrode for 200-400 µsec. The stimulation electrode
was positioned in the molecular layer at the surface of the slice, and
stimulus intensity was increased until IPSCs were evoked.
Paired recordings. In paired recordings the electrical
activity of the presynaptic interneuron was monitored in the
cell-attached mode, whereas postsynaptic signals were obtained using
whole-cell recording. Experimental procedures for paired recordings as
well as basic properties of IPSCs in 20 connected pairs are described in a separate publication (Kondo and Marty, 1998). In the present paper
we show the results of latency analysis using the same recordings as
data base.
Analysis of spontaneous IPSCs. Synaptic currents were
identified using a detection program, as detailed elsewhere (Vincent and Marty, 1993). Individual synaptic currents were inspected to reject
occasional EPSCs, on the basis of their specific kinetic properties
(Llano and Gerschenfeld, 1993). Double IPSCs were identified by visual
inspection; amplitudes and time intervals for doublets were measured
using cursors.
To calculate the distribution of amplitude ratios for consecutive
events, A2/A1,
assuming random superimposition of the events (see Fig.
2E), single IPSCs were chosen randomly, and their
amplitudes, ai, were measured. The ratios
ri,j = aj/ai were formed
for all i, j pairs with
i j. The normalized average time course of all events, y(t), was calculated, with
y(0) = 1. For a lag  t between two events,
first events have decayed to y(t) times
their peak when the second event occurs, such that the
A2/A1 ratio for
each i, j doublet (event with index i first) is
(ai y(t) + aj)/ai = y(t) + ri,j.
The distribution of such ratios was calculated for each bin (see Fig.
2A), taking as t the central interval
value in the bin, and was normalized to the number of events expected
per time interval bin. The components for each of the bins were then
added together (see Fig. 2E, dotted
curve).
Analysis of the synaptic responses (extracellular
stimulation). For each sweep, IPSCs were detected within a time
window of ~3 msec, using a routine with a threshold amplitude of 10 pA written by C. Pouzat in our laboratory. When no IPSC was detected
(failures), an amplitude was measured in the same window, at a point
determined arbitrarily. As a rule, the amplitude histogram of evoked
IPSCs did not overlap significantly with that of the background noise measured in this manner. The average failure sweep did not contain any
signal having kinetics similar to that of IPSCs, confirming that events
classified as failures did not include undetected IPSCs. If two events
occurred within the detection window, only the amplitude corresponding
to the larger of the two was registered. The coefficient of variation
(CV) was calculated as CV = (varIPSC varnoise) 1/2/meanIPSC,
where varIPSC and
varnoise are the variance of the IPSCs and of
background noise, respectively, and meanIPSC is
the average amplitude of successful responses. In the figures,
background noise histograms and evoked IPSCs histograms are scaled to
the same maximum. For the analysis of the kinetics of single and
multiple events (see Fig. 8), latencies were measured by hand as the
first point of the IPSC that clearly deviated from the baseline
current.
Detection of doublets in paired recordings and extracellular
stimulation experiments was performed as for spontaneous recordings. However, jitter among IPSC components contributed by different release
sites had to be taken into account in the analysis of spontaneous
recordings. Therefore a threshold for the minimum time separating two
events was taken as 1 or 1.5 msec for the analysis of spontaneous
events, depending on the experiment. For analysis of evoked currents,
there was no need for such a threshold, because only single-site
experiments were examined, and the effective minimum separation between
reliably measured doublets was ~0.5 msec.
Statistical test for event pairing. To test whether events
were correlated for short time intervals, the number of pairs observed for one interval bin, nobs, was compared
with that expected for a Poisson process,
np, on the basis of the mean event
frequency. Typically nobs was larger than
np, indicating correlation. To evaluate
the statistical significance of the difference between nobs and np, the
probability was calculated to obtain a number of observations equal to
or larger than nobs, assuming Poisson statistics with mean np. This is:
If p was <0.05 it was considered that there was a
significantly larger number of pairs than predicted for a Poisson
process.
For the analysis of the second component of doublets (intervals of
3.5-19.5 msec) in Figure 2, the same method was used, but the bins
were added together.
Blockers of glutamatergic receptors. For the
experiments on evoked IPSCs using extracellular stimulation,
6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) (10 µM; Tocris Neuramin Limited, Bristol, UK) and
D()-2-amino-5-phosphonopentanoic acid (APV) (50 µM; Tocris Neuramin Limited) were added to the bath to
block excitatory synaptic transmission and to ensure that the observed
IPSCs were evoked monosynaptically. NBQX and APV were omitted in the
study of spontaneous IPSCs and in paired recordings; however, in these
experiments EPSCs could readily be identified on the basis of their
fast decay kinetics (Llano and Gerschenfeld, 1993) and were eliminated
from the analysis.
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RESULTS |
Evidence for multivesicular release in recordings of
spontaneous IPSCs
By analogy to the situation found at the neuromuscular junction,
low receptor occupancy was implicitly assumed in the early formulation
of the one-site, one-vesicle hypothesis (Triller and Korn, 1982).
However, if one release event leads to a high degree of occupancy of
postsynaptic receptors, the signal elicited by a second vesicular
release would be reduced in amplitude, perhaps to the point of being
undetected. Therefore, it is essential to know the degree of saturation
of postsynaptic receptors after one release event to predict the
effects of multivesicular release on synaptic transmission. An
effective method for assessing the degree of saturation of postsynaptic
receptors is to examine closely timed pairs of synaptic currents and to
study in these pairs the relation between the amplitude of the second
event and time interval (Tang et al., 1994). We have recently applied
this approach to bursts of mIPSCs induced by applications of low
concentrations of  -latrotoxin, and we concluded that the degree of
occupancy of postsynaptic receptors after the release of a vesicle is
high at interneuron-interneuron synapses (Auger and Marty, 1997; also see Nusser et al., 1997). If such is the case, multivesicular release
would be revealed in IPSC recordings as closely successive events that
do not summate linearly.
Correlations between consecutive IPSCs were examined in a series of six
cells, where mean event frequencies ranged from 1 to 7 Hz. The number
of events analyzed per cell ranged from 270 to 1191. Figures
1 and 2
illustrate the results for one of these cells. Inspection of the traces
suggested that IPSCs were not independent of each other (Fig. 1). Many
doublets were observed with short intervals (1-5 msec) between peaks.
In most cases the peak amplitude of the second IPSC matched closely
that of the preceding event. Furthermore, the IPSC profiles in the peak
region were very similar for the first and second event of a pair, as illustrated for three different doublets in Figure
1A. The normalized slope of current decay (i.e., the
slope divided by the peak current amplitude) was large for the first
pair (0.276/msec for the first event and 0.237/msec for the second),
low for the second pair (0.005/msec and 0.019/msec), and intermediate
for the third pair (0.084/msec and 0.095/msec), and in each case the
slopes were consistent within a pair. For longer time intervals (up to
50 msec), it was also clear that successive events were often coupled. In this time range bursts of events with homogeneous amplitudes were
observed (Fig. 1B, top trace); nonlinear
summation of the IPSCs indicating saturation of postsynaptic receptors
was evident for time intervals below 20 msec (Fig.
1B, bottom traces). The distribution of
interevent intervals could not be fitted with a single exponential, as
would be expected from a Poisson process, but required three
components. Two of these components are apparent in the plot of Figure
1C, and the third is illustrated in Figure 2A.
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Figure 1.
Multiple IPSCs in interneurons. A,
Examples of closely spaced (<5 msec) IPSCs from an interneuron. Three
double events are shown with two different time and amplitude scales to
illustrate both the overall time course of the events and blow-ups of
the peak region. Note differences in time course and amplitudes among
pairs, and the match between peak amplitudes and time courses near the
peaks within pairs. B, Further examples of repetitive
IPSCs from the same recording. Here several events of similar
amplitudes are separated by intervals of 5-50 msec. C,
Distribution of intervals between successive events. The histogram (bin
size: 10 msec; total duration of the recording: 7.5 min) is fitted with
the sum of two exponentials with time constants of 20 and 357 msec. (A
third, very fast component corresponding to double events such as
illustrated in A is not displayed here.) Initial
amplitudes for the fast and slow components are in a ratio of 1.2:1.
Insert, Initial part of the interval distribution. The
dotted line shows the slow exponential with a time
constant of 357 msec.
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Figure 2.
Analysis of event amplitudes in doublets. Same
recording as illustrated in Figure 1. A, Histogram of
occurrence of doublets for short interevent intervals. Dotted
line indicates the number of doublets expected on the basis of
random superimposition of background activity. B,
Schematic diagram showing the current amplitudes measured for the
analysis. A1 and
A2 are measured from the baseline to the
peak of the IPSC, A'2 is measured from the
onset to the peak of the IPSC. C,
A2/A1 as a
function of interevent interval. Most
A2/A1
ratios are closer to the value of 1 predicted by total event occlusion
after saturation of postsynaptic receptors (continuous
line) than to the curve expected on the basis of the summation
of independent events (dotted line; calculated from the
mean decay kinetics of IPSCs). D,
A'2/A1
versus interevent interval. The ratio
A2':A1
rises from a value that is close to 0 for short intervals up to ~1.
Some of the points shown in C are off-scale in
D. Continuous line: time course predicted
for total saturation, calculated from the decay kinetics of IPSCs.
Again, predictions made on the basis of independence (dotted
line) fail to account for the data. E, Histogram
of A'2/A1
ratios for the data shown in B. The dotted
line represents predictions based on random superimposition of
independent events (see Materials and Methods).
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Three different mechanisms could explain a coupling between release
events: (1) synchronous firing of presynaptic interneurons, (2)
repetitive firing of interneurons at high frequency, and (3) delayed
release of vesicles after one action potential. The first and strongest
argument against synchronous firing of presynaptic interneurons is that
it does not explain the finding that within a pair, the second IPSC is
systematically smaller than the first. In addition, IPSC pairs are
unlikely to result from synchronized excitatory inputs to different
interneurons, because they can be demonstrated in the presence of
blockers of ionotropic glutamate receptors, as exemplified by the
results of Figure 6 below. An alternative source of synchronization
could be electrical coupling, because an electron microscopy study
reported the presence of gap junctions between interneuron dendrites in
the molecular layer of rat cerebellum (Sotelo and Llinas, 1972).
However, paired recordings in neighboring interneurons indicate a lack
of firing synchronicity and a very low occurrence (2%) of electrical
junctions (Vincent and Marty, 1996; Kondo and Marty, 1998). Altogether,
the possibility of synchronous firing of presynaptic interneurons
(mechanism 1) can be dismissed.
The distribution of interevent intervals in the range of 0-20 msec
revealed two categories of doublets (Fig. 2A). There
was first a large number of doublets in the first bin (intervals of 1.5-3.5 msec, corresponding to events such as those of Fig.
1A). In this bin, the number of pairs was 21, 3.6 times higher than predicted assuming superimposition of randomly
occurring background IPSCs (dotted line). The probability of
obtaining a number of double events of 21 or larger assuming a Poisson
process given the mean IPSC frequency observed in this experiment can
be calculated as 9 × 107 (see
Statistical test for event pairing in Materials and
Methods). Therefore, the amplitude of this first bin demonstrates a
very significant degree of coupling for events at intervals of <3.5 msec. This was followed by a series of bins (intervals 3.5-19.5 msec;
pairs corresponding to events such as those in Fig.
1B) where the frequency was consistently higher (by
1.2-to 2.1-fold) than that predicted on the basis of background
activity. Again the probability that these numbers could be obtained on
the basis of a Poisson process was very low (p < 0.0002). The maximum firing frequency of cerebellar interneurons is
near 100 Hz at room temperature (Midtgaard, 1992; C. Pouzat, personal
communication), indicating that the duration of their refractory period
is on the order of 10 msec. Therefore, the early component (intervals
of 1.5-3.5 msec) occurs during the refractory period of the
presynaptic spike and corresponds to delayed release after one action
potential (mechanism 3). On the other hand the excess events at 5-20
msec could be caused by either delayed release or repetitive
presynaptic firing. Bursts of IPSCs were previously observed at
interneuron-Purkinje cell synapses, indicating that interneurons
occasionally fire at high rate (Vincent et al., 1992; Vincent and
Marty, 1996). Multiple IPSCs with intervals in the 5-20 msec range
often appeared as bursts with homogeneous amplitudes and regularly
spaced time intervals (Fig. 1B), indicating that they
were induced by repetitive firing of interneurons at high frequency
(mechanism 2).
For intervals between 1 and 3 msec, the frequency of doublets was on
average 5.0 ± 1.9 times higher (mean ± SEM;
n = 6) than predicted on the basis of independence. In
five out of six cells, the deviation from independence was significant
at the p < 0.02 level. On average, however, the
proportion of such doublets was rather small (range, 1.4-2.4%;
n = 5; percentages corrected for background
activity).
Mean ratios of doublet frequencies over values predicted on the basis
of independence for intervals of 5-20 msec were 1.5 in the case of
Figures 1 and 2, 1.7 in another cell, and close to 1 (range, 0.77-1.1)
in the four other cases. Thus in four out of the six analyzed
recordings, repetitive presynaptic firing was not sufficiently
prevalent to give a detectable excess of doublets in this time
window.
In summary, the results of Figures 1 and 2 show that spontaneous IPSCs
in cerebellar interneurons are not randomly interspaced and that there
is a significant pairing of events of similar sizes. In most
experiments multiple release events occur at very short intervals,
reflecting at least in part multivesicular release, whereas in some
experiments, additional doublets with larger intervals reflect
repetitive firing of the presynaptic cells.
Nonlinear summation of closely spaced spontaneous IPSCs
A quantitative analysis of spontaneous IPSC amplitudes for time
intervals shorter than 20 msec is shown in Figure
2B-E. Amplitudes were labeled as illustrated in the
diagram of Figure 2B. Baseline-to-peak (A2) and onset-to-peak
(A'2) amplitudes of the second IPSC were divided by the baseline-to-peak amplitude of the first IPSC
(A1) to compare the two events in a
doublet. Figure 2C shows that for most of the doublets with
an interevent interval between 0 and 5 msec, the ratio
A2/A1 is
approximately 1. On the other hand, the ratio
A'2/A1 (Fig.
2D) is very small for short intervals and increases
for larger interval values. The distribution of
A2/A1 ratios
displays a broad component and a pronounced peak centered near 1 (Fig.
2E). A simulation of the distribution of
A2/A1 ratios (see
Materials and Methods) assuming linear summation of randomly occurring
IPSCs fitted both the shape and the amplitude of the broad component
(Fig. 2E, dotted line). Therefore, the
0.9-1.2 peak of Figure 2E represents the extra
events attributable to multivesicular release and repetitive
presynaptic firing. Similar results were obtained in the other
recordings. For intervals of 0-5 msec, the percentage of
A2/A1 values
between 0.8 and 1.2 was on average 73 ± 11% (mean ± SEM;
n = 6). For comparison, the percentage predicted on the
basis of independence from the amplitude distribution in the experiment
illustrated in Figures 1 and 2 is 19%.
The nonlinear summation of closely spaced release events evident in
Figure 2D is similar to that reported earlier for
-latrotoxin-induced IPSCs and could result from partial receptor
saturation for events occurring at the same synaptic sites.
Alternatively, the smaller second events could be caused by the
liberation of a single vesicle at a site that has not released, whereas
the first event would reflect the synchronized release of several
vesicles at other release sites. If the second hypothesis was correct,
the mean size of the second event in a pair should not depend strongly on the interevent interval and should be close to the mean amplitude of
mIPSCs. Contrary to these predictions, the mean value of the second
event amplitude, A'2, clearly grew for
intervals between 1 and 5 msec (Fig. 2D).
Furthermore, the mean of A'2 values at intervals
of 1-3 msec (26 pA in the experiment illustrated) was markedly smaller
than that of mIPSC amplitudes measured in tetrodotoxin (141 pA) (Llano
and Gerschenfeld, 1993). This indicates that amplitude differences
within doublets reflect at least partially receptor saturation after
multiple release events at identical release sites, rather than
asynchronous release at different sites.
A third explanation for amplitude occlusion could be that a first
release event causes a local depolarization in the dendrite receiving
the synaptic input and thus reduces the amplitude recorded for the
second event, without necessarily implying receptor saturation. At the
age investigated, interneuron dendrites have a simple morphology with a
maximal length of ~50 µm and a diameter of ~1 µm (Llano et al.,
1997), which is not expected to give rise to serious voltage escape
associated with the sizes of synaptic signals recorded here.
Nevertheless, the possibility of a nonlinear summation of IPSCs
attributable to voltage-clamp errors was investigated. To this end, we
measured evoked IPSCs as a function of membrane potential in control
conditions and in the presence of 2 µM bicuculline, a
dose that induced a reduction of the mean current amplitude to 20% of
the control. Because voltage gradients in dendrites should be
proportional to the current flow, voltage-clamp errors should be
manifest as an alteration of the voltage dependence of the IPSC peak
amplitude on addition of bicuculline. The ratio of the mean amplitudes
at 30 mV to those at 60 mV was 0.526 ± 0.022 (n = 6) in control and 0.517 ± 0.017 (n = 4) in 2 µM bicuculline. Thus,
halving the driving force resulted in a current reduction by nearly
twofold, and the current ratio for the two potentials was the same in
the control and in bicuculline. We conclude that voltage gradients in
the dendrites cannot be the cause of the very strong reduction of the
amplitude observed for short intervals.
Extracellular stimulation of a single presynaptic interneuron: some
interneuron-interneuron connections involve a single release site
To pursue the mechanisms underlying double IPSCs, we next studied
evoked IPSCs at individual interneuron-interneuron synapses. Postsynaptic currents were recorded in the whole-cell configuration, and a presynaptic interneuron was stimulated using a pipette filled with extracellular saline and positioned in the molecular layer. Once a
connection was found, the synaptic responses to various stimulus
intensities were recorded. If a single presynaptic neuron is
stimulated, both the probability of obtaining a synaptic current and
the mean amplitude of IPSCs (both excluding and including failures)
should exhibit a clear threshold without further changes with
increasing stimulus intensity (Raastad, 1995). Results were inspected
visually during the experiments to judge whether these requirements
were fulfilled, and if they were not, new presynaptic locations and, as
needed, new postsynaptic cells were assayed. Confirmation that the
response-intensity curve was stepwise was obtained later by off-line
analysis (Fig. 3A,B). Some
unitary synaptic connections showed a very simple amplitude
distribution with two clearly separated groups of events: one group
corresponding to the failures and the other corresponding to successful
responses (Figs. 3C, 4).
Simple amplitude distributions could generally be well fitted by a
single Gaussian (Figs. 3C, 4; however see Fig. 7). Unless
specified otherwise, only connections exhibiting simple distributions
such as that of Figure 3C were used in the present study.
These connections will be called hereafter "simple connections" or,
for reasons that will be discussed below, "single-site connections."
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Figure 3.
Single-site IPSCs elicited with external
stimulation of a presynaptic interneuron. A, Fifty
consecutive sweeps (except for 3 sweeps that were excluded because of
contamination by background IPSCs) showing currents evoked by
extracellular stimulation (5 V stimulus intensity). The time of the
voltage step applied through the stimulation pipette is indicated by an
arrow. Stimulation frequency is 1 Hz. An average failure
sweep was subtracted from all displayed traces. Note that many
stimulations result in transmission failures. B, Plot of
mean amplitude (±SEM) versus stimulation intensity. The synaptic
response has a sharp threshold for stimulations between 2 and 3 V, and
there is no further increase with increasing intensity, indicating that
there is no further recruitment of presynaptic connections. Such a
stepwise dose-response curve is one requirement to ensure that a
single presynaptic neuron is stimulated. C, Amplitude
distributions for stimulations that elicited postsynaptic responses
(bin size 10 pA) and for the failures (bin size 1 pA) (stimulus
intensity: 5 V). The failures distribution was scaled to the
distribution of IPSCs to allow comparison of the SDs. A Gaussian fit of
the responses distribution is superimposed (thick line).
n = 350 stimulation trials. The probability of
successful responses was 32%; mean amplitude, 66 pA; CV (corrected for
background variance), 13%. This type of distribution most likely
corresponds to a single release site.
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Figure 4.
Effect of lowering the external calcium
concentration at a single-site connection. A, Fifty
consecutive sweeps in the presence of 2 mM
Ca2+ and 1 mM Mg2+
(left), and 1.5 mM Ca2+
and 1.5 mM Mg2+ (right).
Successful responses are less frequent and have more homogeneous
amplitudes in the low Ca2+ solution.
Arrowheads indicate stimulation timing. An average
failure sweep was subtracted for display. B, Amplitude
distributions. Left, CTL, 2 mM Ca2+ and 1 mM
Mg2+. n = 500; proportion of
failures: 65.8%; mean amplitude of responses (excluding failures):
131 ± 26 pA; CV = 19.7%. Middle, 1.5 mM Ca2+ and 1.5 mM
Mg2+. n = 200; proportion of
failures: 83%; mean of responses: 120 ± 11 pA; CV = 9.2%.
Right, Wash, n = 200;
proportion of failures: 58.7%; mean of responses: 122 ± 24 pA;
CV = 19.7%. C, Mean amplitude of responses
(excluding failures; each point corresponds to 100 stimulation trials)
versus time. The line is a linear fit of the control data; its negative
slope reflects a slow rundown of the responses. In the presence of 1.5 mM Ca2+, the amplitude decreases
slightly below the regression line, and the SD decreases by ~50%.
Both effects are reversible.
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At simple connections, the mean amplitude of evoked IPSCs (excluding
failures) varied among experiments between 30 and 298 pA; the average
across these means was 101 ± 74 pA (mean ± SD; n = 19; the corresponding CV value was 73%). This mean
is similar to the average for mIPSC amplitudes (141 pA) (Llano and
Gerschenfeld, 1993). The associated CV is also similar to that
calculated from the variations among mIPSC amplitudes recorded in a
given cell (e.g., 82% in the example in Auger and Marty, 1997, their
Fig. 1). On the other hand, at individual simple connections, the CV of
IPSCs excluding failures ranged from 10 to 31%, with an average of
19 ± 6% (mean ± SD; n = 19). These results
indicate that although quantal sizes vary markedly among different
simple connections, they are quite homogeneous for a given
connection.
The probability of getting a response in simple connections was on
average 28 ± 12% (mean ± SD; n = 19),
varying between 10 and 54%.
Recent results at excitatory synapses indicate that amplitude
distributions of miniature (Forti et al., 1997) and evoked synaptic currents (Gulyàs et al., 1993; Arancio et al., 1994; Silver et al., 1996) at single release sites are Gaussian. Therefore the shape of
the amplitude distribution of Figure 3C suggests that the
corresponding synapse involves only one release site. This proposal is
consistent with the low success rate observed at simple synapses,
because the probability of release at single release sites is usually
assumed to be below 0.5 (Raastad et al., 1992). Further support
in favor of the single-site hypothesis comes from a
comparison between IPSCs recorded at simple synapses and bursts of
mIPSCs resulting from application of low doses of
-latrotoxin (Auger and Marty, 1997). Like those observed at simple
synapses, amplitude distributions for -latrotoxin-induced bursts are
Gaussian, with mean values that vary widely among bursts (range,
14-195 pA). Insofar as -latrotoxin-induced bursts originate at
single release sites (Auger and Marty, 1997), this similarity supports the view that the same applies for simple synapses.
Nevertheless alternative interpretations can be proposed. It is thus
possible that several sites contribute to the response peak of simple
connection amplitude histograms and that they cannot be distinguished
because they have similar quantal sizes. In such a case, simultaneous
release from two of these sites should generate a secondary peak with
twice the amplitude of the first. For example, if one assumes two
independent sites with equal release probabilities, the amplitude of
the secondary peak is expected to be 10.6% of that of the first in the
case of Figure 3C; no evidence for such a component is
apparent. Similar arguments can be made for more than two release sites
or for release sites with different release probabilities. Therefore it
is unlikely that several independent release sites generate the
histogram of Figure 3C.
Next, the possibility of several coupled release sites was considered.
At some mossy fiber-granule cell synapses, EPSC amplitude histograms
display a single peak with low variance attributable to the
simultaneous activation of several release sites (Silver et al., 1996).
Such a configuration is unlikely in the present case because of the
high failure rate. However, it could be considered as a possible model
for simple synapses if most failures were caused by stimulation or
propagation failures, whereas responses would correspond to the
simultaneous activation of several sites having a very high release
probability. In granule cell synapses, the existence of multiple
release sites was revealed by lowering the external
Ca2+ concentration, which induced a reduction of the
mean amplitude of responses, excluding failures (Silver et al., 1996).
As will be documented next, very different results were obtained in
interneuron-interneuron synapses, such that the hypothesis of several
coupled release sites could be dismissed.
Lowering the probability of release at simple synapses
The probability of release was reduced by lowering the external
Ca2+ concentration to see how this would affect the
amplitude histograms of the responses. Lowering the external
Ca2+ concentration to 1 mM or less often
led to very low response probabilities that could not be analyzed
quantitatively. The Ca2+ concentration was therefore
usually lowered to 1.5 mM. The external Mg2+ concentration was increased to maintain the
total concentration of divalent ions constant and to limit
modifications of surface potential attributable to ionic changes.
Figure 4A shows the effect of lowering the external
Ca2+ concentration from 2 to 1.5 mM on
the distribution of a simple connection. The probability of release is
reduced by 50%, from 34 to 17%. The mean amplitude of the responses
(excluding failures) is minimally affected, with a slight decrease from
131 to 120 pA. Quite importantly, there is no fragmentation of
the initial peak into several smaller amplitude components. Rather,
consecutive events have more homogeneous amplitudes in the low
Ca2+ period than during controls (Fig.
4A,B). This translated into a strong and reversible
reduction of the CV from 19.7 to 9.2% (Fig. 4C).
In contrast to the results illustrated in Figures 3 and 4, amplitude
distributions at "complex" unitary connections are broad and
skewed, presumably corresponding to multisite connections. In the
example of Figure 5, the mean response
amplitude (excluding failures) decreased from 283 to 226 pA after the
external Ca2+ concentration was lowered to 1.5 mM, whereas the CV increased from 53 to 64%. The mean
amplitude was further reduced (to 165 pA) after the
Ca2+ concentration was lowered to 1 mM.
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Figure 5.
Effect of lowering the external
Ca2+ concentration at a multisite connection.
A, Control amplitude distribution. n = 1400; probability of failures: 17%; mean amplitude: 283 ± 151 pA; CV = 53.4%. B, Distribution in the presence of
1.5 mM Ca2+, 1.5 mM
Mg2+. n = 800; probability of
failures: 35%; mean amplitude: 226 ± 146 pA; CV = 64.4%.
Note that for a decrease of release probability of only 25%, the
distribution is shifted to the left, the mean amplitude decreases by
20%, and the CV increases by 20%. C, Distribution in
the presence of 1 mM Ca2+, 2 mM Mg2+. n = 300;
probability of failures: 44%; mean amplitude: 165 ± 83 pA;
CV = 50.4%.
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On average, reducing the external Ca2+ concentration
from 2 to 1.5 mM (increasing the Mg2+
concentration from 1 to 1.5 mM) at simple connections
decreased the probability of responses to 65 ± 18% of the
control value (mean ± SD; n = 5). In 1.5 mM Ca2+, the average amplitude of the
responses (excluding failures) and the CV decreased to 90 ± 6 and
72 ± 18%, respectively, of the control (mean ± SD;
n = 5). Both decreases were statistically significant
(p < 0.05). If the responses were coming from
several release sites with a very high probability of release, lowering the probability of responses should sometimes desynchronize them, and
smaller amplitude responses should be observed, causing a dramatic
increase in CV. There was no evidence for such a fragmentation of the
initial peak, confirming that the responses come from a single release
site.
Changing the external Ca2+ concentration
alters the excitability of neurons such that a diminution of
Ca2+ is expected to increase the rate of firing of
presynaptic cells (Frankenhaeuser and Hodgkin, 1957). Although the
effect on firing threshold would run counter to the observed increase
of the failure probability, it seemed desirable to reduce the release
probability at single-site synapses without altering the external
Ca2+ concentration. Preliminary tests indicated that
5 µM Cd2+ blocks ~50% of the
Ca2+ currents recorded in the soma of interneurons
(n = 3; data not shown). Because the probability of
responses is already very low in control conditions at single release
site synapses, it was decided to reduce the release probability by
adding only 2.5 µM Cd2+ to the
external medium. On average, the probability of responses was decreased
to 60 ± 25% of the control (mean ± SD, n = 7). The block was variable from connection to connection, varying from 4 to 74%. On average, the mean response amplitude and CV were little
affected by the addition of Cd2+; they were 93 ± 15 and 96 ± 29%, respectively, of the control (n = 7). Neither of these slight reductions was
statistically significant. Again, no fragmentation of the amplitude
distribution was observed, indicating that the responses do not result
from synchronized release of vesicles at several release sites but come
from a single functional release site. In some cases, the CV was
significantly reduced, but this was not observed as regularly as when
the external Ca2+ concentration was lowered.
Altogether these results exclude the possibility that simple synapses
involve several release sites with high release probabilities. Therefore we conclude that such connections involve single functional release sites.
An explanation for the reduction in amplitude and CV observed in some
experiments (particularly when reducing the extracellular Ca2+ concentration) is suggested by the above
conclusion that several vesicles can be released at a single site. In
such a case, decreasing the probability of release should result in a
decrease of the mean number of vesicles contributing to each IPSC. This
should result in a reduced amplitude. As the proportion of
multivesicular IPSCs is reduced, the proportion of events involving
single vesicular release is expected to increase. Thus one component of
the variance of the IPSC amplitudes is gradually suppressed, and this
could account for the decrease in CV observed in some cases. However, the expected effects on mean amplitude and CV are modest if the occupancy of the postsynaptic receptors is high. In addition, the
extent of these effects depends on the mean occupancy of the receptors
and mean number of released vesicles per spike in control conditions.
Because such parameters are likely to be specific for each release site
(Auger and Marty, 1997), results are expected to vary widely from one
experiment to the next.
Multivesicular IPSCs at single-site
interneuron-interneuron synapses
We next examined whether multiple events similar to those obtained
in spontaneous IPSC recordings could be detected in evoked IPSCs
obtained at single-site connections. A typical recording is illustrated
in Figure 6. The analysis reveals a
number of doublets for intervals up to 20 msec (Fig.
6Aa,B). Only clear-cut doublets, for which two peak
amplitudes could be measured unambiguously (Fig.
6Aa), were considered for the plots of Figure
6B-D. Additional traces suggested the occurrence of
multiple events, which however could not be unambiguously resolved
(Fig. 6Ab). In the 0.5-1.5 msec bin, the number of
doublets is 92 times higher than that expected from the level of
background synaptic activity (Fig. 6B, dotted
line), so that contamination of the results by signals coming from
other synapses could be neglected. A clear excess of doublets occurs
for intervals up to 5 msec. In this interval range, most of the
doublets display an
A2/A1 ratio
between 1 and 1.5, indicating event occlusion and high occupancy of the
postsynaptic receptors (Fig. 6B-D).
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Figure 6.
Analysis of doublets for a single-site synapse.
Data from a single-site connection (same experiment as in Fig. 4;
results with control external solution) were analyzed as in Figure 2.
Aa, Some IPSCs are followed by a secondary event.
Measured values of A1 and
A2 are indicated by short horizontal
lines. Ab, Many events display an inflection
point (arrowheads) in their rising phase. The two lower
traces in Ab illustrate traces with a shallow minimum
occurring 1-2 msec after the main current transition, indicating
unresolved late vesicular release. All traces in A are
taken from a sequence of 111 sweeps, during which a total of 51 responses were recorded. Of these, seven contained the doublets shown
in Aa, and eight contained unresolved multiple events as
shown in Ab. The timing of extracellular stimulations is
indicated by arrows. B, The frequency of
doublets decreases abruptly with intervals up to 5 msec. Dotted
line, Frequency of doublets expected from random
superimposition of evoked IPSCs with background IPSCs.
C,
A2/A1
ratios are mainly between 1 and 1.5, particularly for intervals shorter
than 5 msec. D, Histograms of
A2/A1
ratios, both for the entire interval range (0-20 msec, open
bars) and for intervals <5 msec (shaded
bars).
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Extracellular stimulation experiments can lead to the uncontrolled
release of neurotransmitters and damage of surrounding cells (Hamann
and Attwell, 1996). Although it is unclear how such changes could have
produced results such as those of Figure 6, it seemed important to show
that the same results could be obtained without resorting to
extracellular stimulation. We have recently described paired recordings
of connected presynaptic and postsynaptic interneurons (Kondo and
Marty, 1998). On the basis of IPSC amplitude distributions, it was
found that ~50% of the interneuron-interneuron connections are
multisite, and that the other half has primarily one site, which is
contaminated by slow currents of unknown origin in ~25% of the cases
and is free from such contamination in the remaining 25%.
Multivesicular IPSCs were clearly apparent in the last category of
paired recordings, thus showing that the results in Figure 6 are not an
artifact of extracellular stimulation. Of a total of seven single-site
synapses that were examined (three with paired recordings and four with
extracellular stimulation), six showed results similar to those of
Figure 6, with a significant (p < 0.02) excess
of doublets for intervals <3 msec over the number expected from the
background rate of spontaneous IPSCs. The mean value of the
A2/A1 ratio varied
among these experiments between 1.17 and 1.45, with an overall mean of
1.30. By using Equation 3 in Appendix , it is possible to calculate
the occupancy of postsynaptic receptors from this ratio. The result,
0.70, is close to the mean value of 0.76 derived from the analysis of
-latrotoxin-induced bursts (Auger and Marty, 1997). In these
experiments the percentage of resolvable doublets, with an estimated
time resolution of 0.5 msec (see Materials and Methods), varied between
3 and 6% of the successful responses and had a mean of 4.3%.
The lack of linear summation of the IPSCs in doublets indicates that
they activate the same set of receptors and come from the same release
site. Overall the results indicate that more than one vesicle can be
released at a single site in response to a single action potential.
Resolution of monovesicular and multivesicular events in certain
single-site recordings
"Simple" or "single-site" synapses have been defined as
synapses where amplitudes of successful IPSCs were grouped in a single component. In some of these recordings it was possible to further distinguish two closely spaced amplitude levels. One example is shown
in Figure 7A. The amplitude
histogram from these data were better fitted with a double Gaussian
than with a single one (Fig. 7B) (maximum likelihood ratio
test; p  0.001). To test whether the two levels could
have resulted from a drift in the mean amplitude of IPSCs, the
recording was split into two ranges of equal duration. The bimodal
appearance of the amplitude histogram and the positions of the
amplitude levels were conserved in the two consecutive data periods
(Fig. 7C), arguing against a drift in the quantal size.
Further support for the existence of two classes of events was given by
the observation of double events jumping from one level to the other
(in which case the only value that was entered in the amplitude
histogram was the higher one), as well as of inflection points in the
rise phase of the larger events near the peak amplitude of the smaller
component (Fig. 7A).
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Figure 7.
A single-site synapse with two closely separated
amplitude components. A, In this single-site recording,
two distinct amplitude levels were observed. In several traces, double
events were seen to jump from one level to the other (thick
line responses), or to display an inflection point near the
lower amplitude level (arrowhead). B,
Overall amplitude histogram from this experiment (480 trials), showing
two distinct peaks. In dual component traces only the peak amplitude of
the second event was entered. The histogram was fitted to the sum of
two Gaussian curves (thick line; dotted
lines indicate each curve separately) with mean amplitudes and
SD values of 147 ± 14 pA and 198 ± 20 pA, respectively. The
scaled noise histogram is also shown (failure rate was 0.50).
C, Histograms for first (thick line) and
second (dotted line) halves of the data. Although the
proportion of events in the higher amplitude peak decreased from the
first to the second data range, the two peaks appear in both
cases.
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Could the distribution of Figure 7B be generated by two
independent sites? If the amplitudes of the two sites were to
correspond to the positions of the two peaks, one would expect to see
an additional peak with an amplitude corresponding to the sum of the
two components. If we now assume that the larger peak results from the
simultaneous activation of events from the first peak and of events
generated at another site, we would expect to obtain a component at an
amplitude equal to the difference between those of the two peaks.
Because additional components corresponding to the sum or to the
difference of the amplitudes of the two peaks are absent, it appears
that the two levels are not generated by two independent sites. Thus,
the events of the two peaks interact strongly with each other. For
example, if the larger amplitude events result from the summation of
events coming from the first site and from smaller events originating
at a second site, all failures at the first site need to induce
failures at the second site, implying that all failures at the first
site are stimulation or propagation failures, which is an unlikely
hypothesis. Thus, the simplest interpretation of the results is that
all events originate at a single release site, that lower amplitude
events represent single vesicular release, and that the larger ones
represent multivesicular events.
The two-component Gaussian analysis of Figure 7B indicates
that the low and high amplitude components are centered at 147 and 198 pA, respectively. The ratio between these two values is A2/A1 = 1.34, similar to the mean ratio found from the doublet analysis illustrated
in Figure 6. It is again possible to estimate the occupancy of
postsynaptic receptors,  , by using Equation 3 in Appendix . The
result is = 0.66. In two other similar cases the calculated
occupancy values were 0.53 and 0.48. Overall the values of are
somewhat lower than those calculated on the basis of double event
analysis (0.48-0.66 vs 0.55-0.83). Two factors should contribute to
give low values with the present method. First, the large amplitude
peak is likely to contain multiple release events together with
two-release events, leading to an overestimate of the
A2/A1 ratio.
Second, the identification of the two levels is easier for synapses
with unusually high
A2/A1 ratios, and
therefore the method of Figure 7 presumably selects experiments with
low occupancy values.
From the integrals of the two Gaussians in Figure 7 it was calculated
that 61% of the responses corresponded to single release events and
39% to multiple (probably mostly double) events. Thus the mean number
of released vesicles per successful response is at least 2 × 0.39 + 1 × 0.61 = 1.39. The corresponding values for the two
other experiments were 1.38 and 1.13.
The decay of GABA currents after short applications of GABA to
outside-out patches is independent of the GABA concentration (Jones and
Westbrook, 1995; Galarreta and Hestrin, 1997). Therefore, if two
vesicles are released simultaneously, the resulting current is expected
to decay like a single-vesicle IPSC, but if there is jitter among
release events, postsynaptic receptors should be exposed to a longer
effective GABA pulse for a two-vesicle release, and they should respond
with a slower rise time as well as with a slower decay (Jones and
Westbrook, 1995). To test these predictions, the kinetic properties of
the presumed single- and double-vesicle components were compared.
Traces from each class of events were selected on amplitude criteria by
using the two-Gaussian analysis of Figure 7B. In each
category, individual traces were aligned with respect to the time point
corresponding to 25% of the maximum amplitude, and they were then
averaged together. As predicted, the average decay time course of the
larger events was slower than that of the smaller ones (Fig.
8A), although the average rising phase was slower and the peak was more rounded (Fig.
8B). Similar effects were found in the two other
cases of double-peaked single-site histograms. In outside-out patches, increasing the duration of a GABA pulse leads to a slower decay of the
current attributable to an increase in the weight of the slow
component, whereas neither the fast nor slow component time constants
are modified (Jones and Westbrook, 1995). In interneurons, decay
kinetics differs among release sites (Auger and Marty, 1997). Of the
three cases observed, only the connection presented in Figures 7 and 8
had a biexponential IPSC decay. In this case, as predicted from the
above data, the time constants for the fast and slow components were
not modified, and the weight of the slow component was increased for
the large events (Fig. 8). Altogether the results suggest that the
larger events result from the summation of slightly desynchronized
release events.
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Figure 8.
Kinetics of single and multiple events. Same data
as in Figure 7. In A and B, the data have
been split into two components: a low amplitude component, up to 150 pA, and a high amplitude component, for amplitudes larger than 180 pA.
These threshold values were chosen such that each component contains
almost exclusively events from one or the other of the two Gaussians
used to fit the histogram in Figure 7B.
A, Superimposed normalized means of the low
(continuous line) and high (dotted line)
amplitude components. Rising phases of individual events were aligned
before averaging. The decay phases of the high and low amplitude IPSCs
have been fitted using double-exponential curves, with respective
parameters:  fast = 2.6 msec, slow = 12.9 msec; weight of the slow component: 91% (high amplitude);
fast = 3.4 msec, slow = 12.1 msec; weight
of the slow component: 78% (low amplitude). B, Rising
phases of the mean two components without scaling
(a) and after normalization
(b). The high amplitude component has a more
prolonged rise time (+0.2 msec) and a more rounded peak.
C, Mean currents grouped according to latency. The
latencies range from 1.1 to 2.1 msec after the end of the stimulation
artifact in 0.2 msec increments. The earlier latency values correspond
to larger currents (dotted lines), whereas the later
latencies correspond to smaller current amplitudes (continuous
lines).
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Correlation between amplitudes and latencies at
single-site synapses
The results of Figures 7 and 8 suggest that multivesicular events
occur frequently, but that they are difficult to resolve because of a
high degree of synchrony among release events. In such a situation,
multivesicular release at single-site synapses may impose a link
between first latency and peak amplitude of IPSCs. If several events
occur in close succession, a large amplitude is obtained because of
sublinear summation of individual events. In such a case it is the
latency of the first event that is measured, because the analysis
program selects the time point of the first clear deviation from
baseline to calculate the latency. Thus unresolved multivesicular
events are registered as single IPSCs with, on average, large
amplitudes and short latencies. Conversely, late latencies tend to be
associated with single release events, and therefore with small peak
amplitudes, because the probability that a late release would be
followed by another one is low. In agreement with these predictions, we
found in extracellular stimulation experiments at simple synapses that
peak amplitudes are negatively correlated with first latencies (Fig.
8C).
It is conceivable that latency measurements obtained using
extracellular stimulation could be distorted by the unknown delay between stimulation and presynaptic firing. Therefore we also performed
latency analysis on data obtained in paired recordings (Fig.
9). Figure 9A illustrates an
example from a simple synapse. Latency distributions had a single peak
occurring 1-1.5 msec after the peak of the presynaptic spike, and they
trailed up to 2-3 msec after the presynaptic spike. When all data
points were considered, a significant correlation was found between
latency and amplitude (p < 0.01). A regression
line through all data points had a slope of 22 pA/msec. Figure
9B illustrates the results from the experiment that gave the
strongest correlation from a complex connection. In this case the slope
was much larger (502 pA/msec), and the correlation was highly
significant (p < 0.001). Overall 17 pairs out
of 20 showed a statistically significant negative correlation between
the two factors (p < 0.05), and no significant
positive correlation was found. The strength of the correlation was
larger for multisite connections than for single-site connections. This is illustrated in Figure 9C where normalized slopes (i.e.,
slopes divided by the mean current amplitude) are displayed for
single-site connections (left panel; n = 9)
as well as for multisite connections (i.e., for the rest of the paired
recordings) (right panel; n = 11). The
stronger slope obtained at multisite connections is easily explained,
because at these synapses many release events arise at different sites,
in which case their amplitudes add together, whereas at single-site
synapses receptor saturation results in sublinear amplitude
summation.
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Figure 9.
Latency-amplitude correlation in paired
recordings. Results in A and B are from a
single-site and multisite synapse, respectively. a,
Amplitude histograms (failures are not shown). Aa, Mean
current amplitude, 75 pA; total number of trials, 774; failure rate,
0.63. Ba, Mean current amplitude, 476 pA; total number
of trials, 868; failure rate, 0.06. b, Latency
distributions. Latencies are measured from the peak of the presynaptic
action potential-related signal measured in the cell-attached mode. For
each latency bin that contained more than five events, mean amplitude
values ± SEM are displayed. Amplitudes are significantly
correlated to latencies (p < 0.01).
Regression lines have been drawn to the data, with slopes of 22
pA/msec (A) and 502 pA/msec
(B). c, Average traces for latency
bins in b containing more than five events.
C, Summary data for 9 single-site synapses and 11 multisite connections. Some of the single-site synapses were
contaminated with "slow" synaptic currents (Kondo and Marty, 1998).
Aa is reproduced from Kondo and Marty, 1998.
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We have modeled the amplitude-latency correlation at single-site
synapses by assuming a common time-dependent release probability function for all vesicles available for release (Appendix ). Figure
10 shows simulations modeling the data
illustrated in Figure 9A. A first simulation assumed a
time-dependent release probability function that was the same for all
trials. The resulting curves strongly deviated from the data for all
tested values of ( 0.5) (Fig. 10A). Because
other results (Auger and Marty, 1997; Nusser et al., 1997; and present
work) indicate values of in excess of 0.6, we conclude that the
underlying model is inadequate. To obtain enough variation of the mean
amplitude with latency, it was necessary to increase the mean number of
vesicles simultaneously released per trial. We then assumed that the
probability of vesicular release is not constant from one trial to the
next and that for some trials this probability was 0 at all times,
whereas for the other trials it had the reproducible time profile
p(k). With these assumptions the failure rate for
effective trials (those for which p > 0),
F', is lower than the measured failure rate F.
Equations 7-17 are nevertheless valid provided that F is
replaced by F'. Thus m', the mean number of
vesicles per effective trial, is ln F', not
ln F (see Eq. 2). By assuming a value of 2 for
m' (instead of m = 0.462 in panel
A), a good fit was obtained with occupancy values between
0.6 and 0.7 (Fig. 10B). The value m' = 1 gave a good fit only for ~0.5, which was considered too low. The
value m' = 4 gave a shallow variation of the mean amplitude
with latency for the first millisecond, attributable to receptor
saturation, which did not match the experimental data. Therefore the
value m' = 2, although approximate, can be regarded as a
realistic estimate for this experiment, within the framework of the
model exposed in Appendix and 2. m' = 2 corresponds to a
mean vesicle number of 2.31 per successful response.
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Figure 10.
Simulation of latency-amplitude correlation.
Data from the experiment illustrated in Figure 9A.
Simulations were made for a single-site synapse assuming various
degrees of multivesicular release and receptor saturation.
A, In this section the mean number of vesicles released
per trial is 0.462, as calculated from the failure frequency for a pure
Poisson process. The top panel (a)
shows the original latency data (dots; bins of 0.25 msec), a corresponding model latency distribution, and the associated
"driving function" describing the probability density of synaptic
vesicles to undergo exocytosis (Eq. 8; = 0.25 msec; see Appendix
for details). Latencies are shifted by 1.3 msec so that 0 time is the
origin of the driving function. The middle panel
(b) shows the calculated proportions of events
corresponding to the fusion of one, two, or more than two vesicles as a
function of latency. Note that the fraction of events with
multivesicular release drops very quickly with time. The bottom
panel (c) shows simulations of the mean
amplitudes as a function of latency, assuming occupancy values of 0.5, 0.6, 0.7, and 0.8. None of these simulations comes close to the
experimental data (dots; error bars show ± SEM).
B, For these simulations the density probability of
vesicular release was allowed to fluctuate between 0 for some of the
trials and a constant driving function in the other trials. This
introduced one more free parameter than in the simulations shown in
A. For the simulation shown here the mean number of
vesicles that was chosen was two. a, b,
and c are arranged as in A. Occupancy
values between 0.6 and 0.7 give a good fit to the data.
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As can be seen from Figure 10, the maximum slope of the
amplitude-latency plot increases with m' and decreases with
. Because m' is different in Figure
10Ac,Bc, it can be seen that for the same occupancy
the slope of the amplitude-latency relation is different. A slope of
20%/msec, corresponding to the mean value for single-site synapses in
Figure 9C, is obtained for = 0.6 and m' = 0.6. Because our estimate of is larger than 0.6, we infer that
m' is on average larger than 0.6. According to the Poisson
statistics exposed in Appendix , this corresponds to a mean vesicle
number of >1.33 per successful response and to a proportion of
multiple events of >27% among successful responses.
| |
DISCUSSION |
Single-site synapses at interneuron-interneuron connections
Multivesicular release can be demonstrated at the level of
spontaneous synaptic activity (Figs. 1, 2), where signals arise from
several synaptic connections and where many connections involve several
release sites. This approach has the advantage that it is readily
applicable to any slice preparation. However, quantitative interpretation of the results is obviously much easier if experiments can be performed at single connections involving one release site. In
another study, we present results from single-site
interneuron-interneuron synapses using paired recordings (Kondo and
Marty, 1998). Some of the results presented here (Fig. 9) were obtained
with this technique. For the major part of the results, however, we
took advantage of the flexibility of extracellular stimulation to
select "simple" connections where successful responses can be
grouped in a single peak. Furthermore, we showed that when the
probability of release is decreased at these junctions, the position of
the mean does not change markedly, and the CV usually does not
increase. These experiments (Fig. 4) dismiss the possibility that the
single peak histograms would arise from several sites with very high release probabilities (Silver et al., 1996). Taken together, the results provide convincing evidence that a fraction of
interneuron-interneuron synapses ("simple" connections) involve a
single functional release site. However, as already discussed in Auger
and Marty (1997), it should be kept in mind that a single functional
release site does not necessarily correspond to an anatomically defined
single release site. Because cross-talk between neighboring sites can occur for distances on the order of 1 µm or less (Clements, 1996; Barbour and Häusser, 1997), two closely apposed release sites with a separation of <1 µm could function as a single one. A recent electron microscopy study indicates that in interneurons of the molecular layer of the cerebellum, the density of GABAergic synapses on
a given postsynaptic dendrite is low (Nusser et al., 1997). Some
closely apposed sites were seen occasionally (Fig.
2A), but in the more general case synaptic contacts
consisted of isolated release sites. This indicates that single
functional sites are likely to correspond to single morphological
release sites.
Evidence for multivesicular release at single release sites
The evidence indicating multivesicular release at single-site
interneuron-interneuron synapses can be summarized as follows.
Event pairs with amplitude occlusion
This is the most direct evidence for multivesicular release. Event
pairs with time intervals of <5 msec are observed in spontaneous synaptic currents, in single-site IPSCs obtained with extracellular stimulation, and in single-site IPSCs obtained in paired recordings. In
all three experimental conditions, the frequency of these events is
much higher than that expected from the superimposition of simple
events with the randomly occurring background synaptic activity. A
further argument against random superimposition of events is that the
amplitudes of second events are on average markedly smaller than that
of the preceding one. This phenomenon, amplitude occlusion, suggests
that the first and second event in a pair share the same postsynaptic
receptors. The finding of similar occupancy values for multiple events
at single-sites synapses (0.70) and in -latrotoxin-induced bursts
(0.76) (Auger and Marty, 1997) is an important check of the internal
consistency of our analysis. Finally it should be noted that the number
of event pairs drops steeply as a function of time interval (Fig.
6B). The time constant of this decay cannot be
determined precisely from our experiments, but it is of the same order
of magnitude as that of the "driving function" derived from first
latency histograms (~0.25 msec) (Fig. 10), as expected from a model
where the release of several vesicles occurs independently of each
other (Appendix ).
Correlation between amplitude and kinetics
At single-site synapses, unresolved multivesicular events are
expected to have larger amplitudes (because of incomplete receptor saturation by a single vesicle), slower rise times (because of jitter
between the constituent elementary events), and slower decays (because
of a more prolonged neurotransmitter concentration transient) than
single events. Accordingly, the multivesicular release hypothesis
predicts a positive correlation between amplitudes and rise times, and
between amplitudes and decay times. Such correlations were found in
single-site synapses both when using extracellular stimulation (results
not shown) and in paired recordings (Kondo and Marty, 1998). These
results are confirmed and further quantified by experiments such as
that of Figure 8, which compare mean kinetics for single and multiple
events at a single-site synapse.
Correlation between amplitudes and latencies
At single-site synapses, amplitudes are correlated to latencies as
expected on the basis of multivesicular release and partial receptor
saturation (Figs. 8-10; Appendix ). A similar effect was described
recently at what may have been a single-site pyramid-pyramid synapse
in a cortical slice (Markram et al., 1997, their Fig. 3).
Separation of amplitude histograms between simple- and
multiple-event components
In certain single-site amplitude histograms two components can be
distinguished within the response peak (Fig. 7). The ratio between
large and small amplitude levels is consistent with the notion that the
small and large amplitude events correspond to single and multiple
vesicular release, respectively.
Multivesicular synaptic currents have not been reported at single-site
synapses before the present work. It is worth pointing out that in
earlier publications (Gulyàs et al., 1993; Arancio et al., 1994;
Silver et al., 1996) the quantal size is on the order of 10 pA, an
order of magnitude smaller than in the present work. The large quantal
size of interneuron synaptic currents is probably the key factor that
allowed us to demonstrate multiquantal events.
Estimating the number of released vesicles at single sites
Because of the fast kinetics of vesicular release and of the high
but incomplete occupancy of the postsynaptic receptors, most of the
multivesicular release events were not detected as such. Therefore the
proportion of doublets that was measured in single-site experiments
(4.3%) must be considered an unrealistic lower estimate of the true
proportion of multiple release events. Two methods can be used to
obtain a better estimate of the proportion of multiple release. The
most direct approach is to measure the proportion of smaller and larger
events in the cases where two subpeaks were visible in the amplitude
distributions. The proportion of multiple release obtained with this
method, 30% on average, suggests that multiple release is in fact a
quite prevalent phenomenon. The same conclusion can be drawn from the
more elaborate analysis of latency-amplitude correlation plots (Figs.
9, 10). For the experiment shown in Figure 10, a proportion of 69% of
multiple events can be calculated. It is likely that this number varies
from one synaptic site to the next, as do other parameters describing
basic properties of synaptic transmission (Auger and Marty, 1997;
Nusser et al., 1997). We argued from the analysis of Figure 10 that
when results are averaged across preparations, >27% of evoked IPSCs
correspond to multivesicular release. Because the release probability
increases steeply with temperature in slice preparations (Hardingham
and Larkman, 1998), and because our experiments were performed at room
temperature, the percentage of multivesicular events is likely higher
at physiological temperature.
Fluctuations in the release probability function from trial
to trial
We have considered two variants for the model of the release
probability of synaptic vesicles. In one variant the probability is
constant from trial to trial, and the numbers of vesicles released per
trial follow a Poisson distribution. In the other variant the
probability is allowed to drop to 0 for some of the trials, as would be
expected, for example, if there were propagation failures. The second
model was required to obtain a reasonable fit of the data in Figure 10.
We stress, however, that the all-or-none alternation in release
probability assumed for Figure 10 was chosen for the sake of
simplicity; other schemes allowing more gradual fluctuations of the
release probability are just as likely. Still other models, assuming
for instance a process by which the release of a first vesicle would
facilitate the release of another one, cannot be excluded. However, the
suggestion that some of the synapses exhibit variations in the release
probability from trial to trial is attractive because it is in line
with previous results obtained at interneuron-Purkinje cell synapses
as well as at interneuron-interneuron synapses (Vincent and Marty,
1996; Kondo and Marty, 1998). Thus evidence is accumulating suggesting
that quantal fluctuations are not the only determinant of variations in
GABA release at interneuron terminals.
| |
FOOTNOTES |
Received Jan. 26, 1998; revised March 18, 1998; accepted April 6, 1998.
This work was supported by a fellowship from the French Ministère
de la Recherche et de la Technologie (C.A.), by a Human Frontier
Science Program fellowship (S.K.), and by the Deutsche Forschungsgemeinschaft (SFB 406). We thank C. Pouzat and P. Vincent for
sharing analysis software, and L. Forti and I. Llano for comments on
this manuscript.
Correspondence should be addressed to Dr. A. Marty, Arbeitsgruppe
Zelluläre Neurobiologie, Max-Planck-Institut für
biophysikalische Chemie, 37077, Göttingen, Germany.
Dr. Kondo's present address: Laboratory for Neural Circuit,
Bio-Mimetic Control Research Center, RIKEN, Anagahora, Shimoshidami, Moriyama-ku, Nagoya, Aichi 463, Japan.
| |
APPENDIX 1 |
AMPLITUDE DISTRIBUTION AT A SINGLE RELEASE SITE:
COMBINING A POISSON DISTRIBUTION WITH PARTIAL RECEPTOR SATURATION
To model the pattern of release probability, we assume a very
large pool of vesicles, each vesicle having a low probability of
release. All vesicles have the same probability to undergo exocytosis,
and there is no interaction between release events. The numbers of
vesicles released for each trial follow the predictions of a Poisson
distribution, such that the probability to observe 0 or j
(>0) vesicles is respectively:
where m is the mean number of released vesicles.
E(0) is the observed number of failures, F, so
that m = ln (F).
A variant of the Poisson model assumes that the release probability is
0 for some of the trials and is constant (p > 0) for the others. Then the release can again be considered as a
Poisson process for the cases where p > 0. The above
equations then become:
|
(1)
|
|
(2)
|
where m' and F', respectively, represent the
mean number of released vesicles and the proportion of failures under
the condition that p > 0.
The mean amplitudes of single, double, and multiple events can be
calculated by assuming progressive saturation of a common set of
postsynaptic receptors. We call the proportion of receptors occupied after one release event. After a first release with mean amplitude A1, a second release event
finds only a fraction (1 ) of the receptors that are free
for activation. Because interevent intervals are very short (<3 msec)
compared with the decay kinetics of the IPSCs, the partial recovery in
the number of available receptors attributable to deactivation during
the interval between the two events was neglected. Therefore the ratio
of the amplitude increment for the second event over
A1 is
A'2/A1 = (1 ). Because of poor event discrimination, most double-release
events are detected as a single event with the total amplitude
A2 = A1 + A'2 (Auger and Marty, 1997). The ratio to the
mean amplitude of truly single events is:
|
(3)
|
Likewise, after j consecutive release events the total
amplitude is:
|
(4)
|
The maximum of this expression, corresponding to very large values
of j, is A1/.
| |
APPENDIX 2 |
PREDICTION OF AMPLITUDE-LATENCY CURVES
For this calculation the time span of the latency histogram (~2
msec) is fragmented into n t increments, and
we call p(k) the probability to observe a release
event during the time interval [(k 1)
t, k t]. Release events are
assumed to be independent, so that p(k) does not
depend on whether events occurred before the kth interval.
t is small enough such that the probability to observe
two events during one interval can be neglected. The sum of all values
of p(k) represents m, the mean number
of vesicles released for one presynaptic action potential:
|
(5)
|
Let l(k) be the probability to observe a
first latency in the [(k 1) t,
k t] interval. l(k) is
the product of p(k) with the probability that no
event occurred until the kth interval, which is:
Therefore:
|
(6)
|
The sum of the l(k) histogram represents the
probability of observing at least one event after a presynaptic
stimulus:
|
(7)
|
where F represents the probability of failures.
We model l by an function:
|
(8)
|
where is a time factor, and C is a constant, which
is chosen such that Equation 7 is satisfied. Examples of time profiles of the functions p(k) ("driving function")
and l(k) ("latency") calculated on the basis
of Equations 6 and 8 are illustrated in the top panels of Figure
10.
To predict the mean amplitude of events having a first latency in the
kth interval, we need to know the probability that
additional vesicular release events will occur after the kth
interval. The numbers of later release events follow a Poisson process
with an expectation value:
|
(9)
|
Therefore the probabilities Vj
(k) to have j vesicles released after the
kth time interval follow the relations:
|
(10)
|
|
(11)
|
Note that the same equations apply to the probabilities
Vj to observe j release events over
the entire latency distribution, that m0 = m and that V0 = exp (m) = F.
The probability to obtain a total of j vesicles released
with a first latency in the kth interval is:
|
(12)
|
At each time interval the mean current amplitude A
(k) is given by the proportion of events with 1, 2, ... j vesicles such that:
|
(13)
|
These equations, together with Equation 4 allow calculation of the
dependence of mean amplitude on latency, starting from the latency
distribution and from the value of . If the probability of release
is allowed to fluctuate among trials between p(k)
and 0, then the same equations apply, except that m and
F should be replaced by m' and F', as
explained in Appendix .
| |
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S. J. Cragg
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Y. Zhao and M. Klein
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S. Kirischuk, J. D Clements, and R. Grantyn
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A. Abenavoli, L. Forti, M. Bossi, A. Bergamaschi, A. Villa, and A. Malgaroli
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T. D. Gover, X.-Y. Jiang, and T. W. Abrams
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V. Scheuss, R. Schneggenburger, and E. Neher
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A. C. Meyer, E. Neher, and R. Schneggenburger
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S. Kirischuk, N. Veselovsky, and R. Grantyn
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C.-L. Zhang, A. Messing, and S. Y. Chiu
Specific Alteration of Spontaneous GABAergic Inhibition in Cerebellar Purkinje Cells in Mice Lacking the Potassium Channel Kv1.1
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O. Prange and T. H. Murphy
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P. J. Mackenzie, G. S. Kenner, O. Prange, H. Shayan, M. Umemiya, and T. H. Murphy
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Top
Abstract
Introduction
